The objective of this core TR&D project is to further improve methods for 3-dimensional immunolabeling, utilizing semithin cryosections for intermediate voltage electron microscopy (IVEM) by addressing issues such as probe penetration, signal enhancement, section quality and contrasting of unlabeled structures. Because of the penetration problems associated with colloidal gold, we have moved away from this approach and focused on other methods. As part of a project on changes in the tranverse tubule system in failing hearts, we have developed a method for performing very precise correlated 3D light and electron microscopy of difficult-to-access antigens using thick cryosections. We have demonstrated that the lectin wheat germ agglutin (WGA) conjugated to a fluorophore can beautifully reveal the structure of the transverse tubule system, but that the penetration of WGA into Vibratome sections of heart muscle is relatively poor. Briefly, this method involves the production of 3-5 5m thick ultracryosections of heart muscle which are collected in special Petri dishes which contain a coverslip incorporated in the bottom (Mat-Tek Corporation) . The Petri dishes are coated with Cell Tak so that the sections adhere firmly to the coverslip. These sections are then labeled with a 50:50 cocktail of WGA-FITC and WGA-HRP. Confocal volumes are obtained through the entire thickness of the cryosection and then the HRP is reacted with diaminobenzidine and embedded for electron microscopy. The embedding process can be performed in the Petri dish, so no further manipulation of the section is required. Following embedding, a series of 1 5m thick sections are obtained through the cryosection and the entire thickness is reconstructed in 3D at the electron microscopic level using serial electron tomography. In this way, the entire thickness of the section is reconstructed at both the light and electron microscopic level, facilitating correspondence of the two resulting volumes. Using this method, we are obtaining quantitative assessments of surface area and volume using both LM and EM to determine the accuracy of such measurements using the confocal and also to obtain estimates of shrinkage caused by dehydration, embedding and exposure to the electron beam. Similar methods are being used to label difficult to access antigens is dense tissues such as muscle using peroxidase-based immunolabeling methods.
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